P467: FUNCTIONAL CHARACTERIZATION OF ABERRANT GATA1 PROTEIN COMPLEXES IN NORMAL AND MALIGNANT HUMAN ERYTHROBLASTS
Authors: S. Tauchmann, F. Otzen Bagger, T. Bock, R. Sivalingam, T. Eder, E. Heyes, A. Fagnan, M. von Lindern, T. Mercher, F. Grebien, J. Schwaller
Journal: HemaSphere (2022)

Background: Terminal differentiation of erythroid progenitor cells is controlled by the master transcription factor GATA1 acting in activating and repressive protein complexes. In acute erythroid leukemia (AEL), cell differentiation is impaired leading to accumulation of immature erythroblasts. Previous work in mouse models and primary human cells suggested impaired function of abundantly expressed GATA1 protein in AEL cells. Interestingly, GATA1 overexpression induced or enhanced terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 erythroblasts, respectively. Aims: We hypothesize that impaired terminal erythroid differentiation in AEL might be the consequence of aberrant GATA1 protein interactions. Methods: We compared the proteomes of three human AEL cell lines (F36P, K562, KMOE2) and primary AEL patient cells with HUDEP2 cells and primary human erythroblasts (hEBST) that were capable of in vitroterminal erythroid differentiation, using a tandem mass tag(TMT)-based approach (n=3/type). In parallel, we compared the GATA1 interactomes by immunoprecipitation (IP) followed by mass spectrometry (MS) (n=3/type). In addition, we performed a targeted CRISPR screen in Cas9-expressing K562 cells in which we monitored growth kinetics upon mutational disruption of candidate genes and simultaneously assessed changes in erythroid differentiation by measuring CD71 and CD235a surface markers. Results: Unsupervised hierarchical clustering displayed a clear separation of AEL proteomes from those of normal erythroblasts with 386 proteins higher expressed in the first and 623 proteins more abundant in the latter group (logFC≥2;q<0.05). GATA1-IP-MS analysis of nuclear lysates from all six cell types identified 1616 proteins, of which 126 were differentially associated with GATA1 depending on cell state. 54 proteins preferably interacted with GATA1 in the AEL group, whereas 72 proteins were more enriched in “normal erythroblasts” (q<0.5). GATA1 interactomes in hEBST and HUDEP2 cells highly overlapped and were more similar to GATA1 partners identified in F36P and KMOE, than those of K562 and primary AEL cells. Integrative analysis revealed 118 proteins that were differentially expressed and/or precipitated by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). To identify if GATA1-associated proteins are involved in erythroid differentiation we functionally characterized them in a CRISPR screen in K562 cells. 4 patterns were observed: inactivation of cluster 1 genes such as ZEB2 or IKZF1 did not change cell differentiation or growth, loss of cluster 2 genes like RPS21 or RANGAP1 did not change differentiation, but impaired cell growth/survival. Disruption of cluster 3 genes like SND1, or EIF5A resulted in an increase in CD71 and/or CD235a expression without significant changes in cell growth, and deletion of cluster 4 genes including SMC1A or ATP1A1 caused significant signs of differentiation together with cell depletion over time. Overall, knockout of 41 genes caused significantly increased CD71 and/or CD235a surface expression, indicating that these GATA1-interacting proteins affect erythroid differentiation. Summary/Conclusion: Our work suggests that impaired erythroid differentiation of AEL cells involves aberrant GATA1 protein complexes. Ongoing gain- and loss of function validation experiments of selected candidate genes and chromatin assays will identify novel regulators of terminal differentiation in normal and malignant erythropoiesis.